A triangulating autofocusing device is known from U.S. Pat. No. 5,136,149 B1. DE 195 37 376 A1 discusses this US patent and refers to the autofocus principle described therein as the “triangulating” autofocus principle. A number of microscopes in the prior art have triangulating autofocusing devices or autofocusing scanner units which use an angular or oblique autofocus measuring beam and a reflecting or regular or directed reflection on the object. Therefore, as shown in the attached FIG. 1, which corresponds to the triangulating autofocus principle known from the above-mentioned U.S. Pat. No. 5,136,149 B1, an autofocusing light source 19 is arranged such that after the deflection of the measuring beam 30 and after said beam 30 has passed through the microscope objective 10 the object plane 16 is struck by the autofocus measuring beam diagonally or at a sloping angle. The autofocusing scanner unit additionally contains a position-sensitive autofocus detector 28 for detecting the lateral shift in the beam (as will be described hereinafter) and a motor 27 for moving the objective 10. Alternatively, the object plane 16 can also be shifted in the direction of the optical axis.
In the autofocusing device with a microscope according to FIG. 1, the measuring beam designated 30 is deflected by the beam splitter 20 at a point A into one half of the beam cross-section (in relation to the optical axis 8). The deflected beam 30 is deflected or diffracted by the objective 10 so as to strike the object plane 16 at a reflection point C at a diagonal or sloping angle. The beam 30 is reflected or sent back or remitted as a reflected measuring beam 32 and is then deflected via the objective 10 once again through the beam splitter 20 at a point B on the other side of the beam path (in relation to the point A). The deflected beam 32 then illuminates the detector 28, e.g. a position-sensitive detector (PSD), the output signal of which is dependent on the location where the beam 32 strikes or makes contact, so that the location is determined in this way.
In the event of defocusing, i.e. in the present instance according to FIG. 1 where the object plane 16 is shifted into the plane 16′ (or a point that is to be imaged is displaced from the plane 16 into the plane 16′), the measuring beam 30 is first reflected at the reflection point D which has been displaced relative to the point C not only in the direction of the optical axis 8 but also laterally or sideways thereto. As shown, the corresponding reflected beam 32′ reaches the detector 28 at a different place and thus delivers a modified signal relative to the focus position. In this way the degree of defocusing can be measured and compensated by the said motor 27 which moves the objective lens.
The following patent specifications deal with systems based on this triangulation principle as described above.
DE 32 19 503 A1 discloses an autofocusing device for optical equipment, particularly reflected-light microscopes. In this apparatus a laser autofocusing arrangement is provided which generates a measuring beam pencil one half of which is screened off by an optical component. The measuring beam pencil which is reduced to half its cross section is coupled into the illuminating beam path of the reflected-light microscope as an autofocus measuring beam and this in turn falls onto an object via the objective pupil and the objective. In this way the measuring beam which is half screened off—preferably pulsed laser light in the IR range—generates a measuring spot on the object for the autofocus which does not interfere with the microscopic observation. During defocusing this measuring sport “migrates” on the surface of the object.
The optical component which covers half the measuring beam pencil may be for example a deflecting prism, in this case, which is half introduced into the measuring beam path up to the optical axis. The side of the deflecting prism pointing towards the laser light source is fully reflective, so that a half screened off measuring beam extends in the direction of the optical axis as far as the objective pupil and is focused on the object through the objective as a measuring spot. After being reflected from the surface of the object the (half) autofocusing measuring beam sent back also extends in the direction of the optical axis back to the said deflecting prism, while during its “return journey” the remitted autofocus measuring beam runs in the pupil half which is opposite to the outward journey, in which the screened off part of the measuring light beam pencil directed towards the object is located. The reflected autofocus measuring beam is conveyed through the deflecting prism to a detector which may consist essentially of a differential diode (two diodes). When the system is optimally focused, the image of the measuring spot is located in a precisely symmetrical position relative to the two diodes of the detector. In the event of defocusing the image of the measuring spot migrates away from the central position in the direction of one of the two diodes, depending on the direction of defocusing. As a first approximation, the amount of displacement of the measuring spot on the differential diode is proportional to the amount of defocusing. The apparatus makes it possible to reverse the detected defocusing by corresponding counter-steering of the objective and/or the stage in the z-direction. With the apparatus proposed therein it is also possible to set defined amounts of defocusing (“offset”) so as to be able to carry out microscopic observations at different heights, for example in the case of objects structured in the z-direction.
An autofocus system with a similar measuring principle is also known from US 2004/0113043 A1. Once again, a half-screened off measuring beam is directed onto an object that is to be examined under the microscope in order to produce a measuring slot. The reflected measuring beam is supplied to a CCD sensor. A signal processing device provided downstream delivers signals for the defocusing to a computing unit (CPU) which in turn controls the stage and/or the objective so as to correct any defocusing. The said measuring slot is generated by means of infrared light, while the image of the measuring slot is reflected at interfaces of the object (surface of the cover glass, surface of the sample underneath the cover glass). The reflected measuring slot is imaged on a line detector (CCD sensor) through optical means which lastly comprise a cylinder lens. The correlation between the corresponding detection signal and the actual focus position is illustrated in the said US patent specification.
A similar autofocusing system for an inverted microscope with transmitted-light illumination is known from U.S. Pat. No. 7,345,814 B2. To minimise the scattered light, a polarisation beam splitter and a λ/4 plate are provided in the beam path of the autofocusing device. In the particular application described therein, the autofocusing device ensures focusing on the cover glass in order subsequently to shift the objective of the microscope by a predetermined amount in the z-direction (“offset”).
For completeness it should be pointed out that an autofocusing device for microscopes is already known from the older German patent specification 21 02 922. A similar device for automatic focusing of a microscope on different object planes is known from Austrian patent AT-353 497.
A feature common to the autofocusing processes described above is that they operate with a fixed half-shutter, particularly a central iris diaphragm, which is switched off on one side from the optical axis to the edge of the beam cross section. As a result the object is illuminated on one side with the autofocus measuring beam (triangulation principle). The result of this geometry, during defocusing, is that the image of the autofocus mark is shifted on the sensor, while the defocusing is proportional to the decentring of the centroid of the image, in a first approximation. The size of the detector delimits the maximum capture range for focus settings in the z-direction in the region of the object. These systems are therefore unsuitable, in particular, for seeking the focus position in the event of a large defocus.
Another disadvantage of the known systems are the so-called first order reflections, which are formed most noticeably at the apices of the optical surfaces (lenses) and have a highly disruptive effect on the measuring signal. The signal-to-noise ratio which is impaired by first order reflections becomes particularly noticeable when an interface between the cover glass and the aqueous solution is used as a reference surface for holding the focus, as the reflection of this interface is only about 4 per thousand (4‰). As a result the autofocus reflection can be covered by the scattered light. To minimise scattered light, therefore, polarisation beam splitters with λ/4 plates are used in the above-mentioned U.S. Pat. No. 7,345,814 B2.
It is therefore desirable to provide an improved triangulating autofocus system for microscopy which avoids the above-mentioned disadvantages of the known prior art as far as possible, and in particular a system according to the invention should have a large capture range, should restrict the influence of disruptive scattered light and/or should be suitable for autofocusing on samples that reflect very poorly.